Date of Award

5-1-2012

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Watabe, Kounosuke

Abstract

Elevated level of lipogenic enzymes and overall lipogenesis have been reported in a wide variety of cancers and blocking the lipogenic pathway by chemical inhibitors or RNA interference causes tumor cell death by apoptosis which provides a strong rationale for targeting lipogenic pathway for the treatment and prevention of cancer however the exact role of lipogenesis as a cause, facilitator or consequence is not yet clearly understood. Therefore in this dissertation research, we set up to determine the mechanism of tumor cell death by inhibiting lipogenesis and to determine the role of increased lipogenesis in the breast cancer progression. In the first part of this study, we investigated the status of fatty acid synthase (FAS) gene which is regarded as the key lipogenic gene in fatty acid biosynthetic pathway and is responsible for the synthesis of lipid molecules by facilitating the condensation reaction between acetyl-CoA and malonyl-CoA in the presence of NADPH. We observed that normal breast epithelial cells MCF10A cells have very low level of FAS expression whereas breast cancer cell lines MCF7, MDA MB231 and MDA MB231 LM have significant overexpression. Next, we observed the similar trend of FAS overexpression in breast cancer stem-like cells (CSCs) isolated from the MCF7, MDA MB231 and MDA MB231 LM cell lines using cell surface markers (CD24-/CD44+/ESA+). These cells were previously transplanted into the mammary fat pad of nude mice and the results of our limiting dilution analysis indicate that CSCs had a significantly higher ability of forming breast cancer in the injected animals which explains our rationale to use CSCs in our research. In order to exploit this lipogenic pathway for the treatment and chemoprevention of breast cancer, we then examined the effects of resveratrol on breast cancer cells. Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. We observed that resveratrol significantly reduced the cell viability by inducing apoptosis in parental cells as well as in CSCs. Resveratrol also inhibited mammosphere formation which is an inherent property of CSCs. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the FAS gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by FAS overexpression suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of CSC in an animal model of human breast cancer xenograft without showing apparental toxicity. Taken together, our results indicate that resveratrol is capable of inducing apoptosis in the CSCs through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol. Taken together, our results indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol. In the second part of research, we tried to determine the role of elevated level of lipogenesis in normal to ductal carcinoma in situ (DCIS) progression. For this, we first analyzed the expression profile of various lipogenic genes using an expression microarray and found that CSCs from DCIS.com showed significantly higher level of ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and FAS than the normal non-tumorigenic stem-like cells obtained from MCF10A. The result was also confirmed by qRT-PCR and Western blot as well as in clinical specimens of DCIS by immunohistochemistry. In the next step, we detected that SREBP1, the master regulator of lipogenic genes, is also upregulated in DCIS and further identified that SREBP1 regulates the co-ordinate expression of ACLY, ACC and FAS ultimately resulting in the elevation of lipogenesis. In order to determine the role of SREBP1 overexpression in normal to DCIS transition, we overexpressed the SREBP1 in MCF10A cells which induced a significant increase in the downstream key lipogenic genes ACLY, ACC1 and FAS which resulted in the clear upregulation of total lipid content in the cells. Furthermore, we found that this elevation of lipogenesis in MCF10A-SREBP1 stem-like cells confers proliferative advantage as well as a significant increase in mammosphere forming ability and anchorage independent growth (3D culture). Thus, our results showed a possibility that increased lipogenesis in normal stem-like cells may be responsible for providing oncogenic transformation properties which can be confirmed at least in our in vitro model. We then examined the effects of resveratrol on CSCs sorted from DCIS.com. We found that resveratrol decreased the cell viability and increased apoptosis by reducing the total lipid content by inhibiting the expression of SREBP1 and downstream lipogenic genes. Resveratrol also hindered the stemness of the DCIS CSCs by inhibiting its mammosphere forming ability. When DCIS CSCs were transplanted into mammary fat pad of nude mice which were on resveratrol treatment, we observed that resveratrol significantly suppressed the formation of DCIS by downregulating lipogenic genes and by upregulating pro-apoptotic genes, DAPK2 and BNIP3. Collectively, our results indicate that lipogenic genes SREBP1 co-ordinately regulates the overexpression of ACLY, ACC1 and FAS in DCIS CSCs at an early stage of breast tumorigenesis and thus confer proliferative and survival advantages. Anti-growth effect of resveratrol on DCIS CSCs also provides us with a strong rationale to use this agent for chemo-prevention against DCIS.

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